The cell in development and inheritance (1900) 7
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Wilson EB. The Cell in Development and Inheritance. Second edition (1900) New York, 1900.
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Chapter VII Some Aspects of Cell-Chemistry and Cell-Physiology=
" Les phenomenes fonctionnels ou de depense vitale aura tent done leur sitge dans U protof las me cellulaire.
" Le noyau est un appareil de synthase org ant que, l' instrument de la production, U germe de la cellule."
Claude Bernard
- 1 eeons sur Us phenomenes de la vie, I., 1 878, p. 198.
A. Chemical Relations of Nucleus and Cytoplasm
It is no part of the purpose of this work to give even a sketch of general cell-chemistry. I shall only attempt to consider certain questions that bear directly upon the functional relations of nucleus and cytoplasm and are of especial interest in relation to the process of nutrition and through it to the problems of development. It has often been pointed out that we know little or nothing of the chemical conditions existing in living protoplasm, since every attempt to examine them by precise methods necessarily kills the protoplasm. We must, therefore, in the main rest content with inferences based upon the chemical behaviour of dead cells. But even here investigation is beset with difficulties, since it is in most cases impossible to isolate the various parts of the cell for accurate chemical analysis, and we are obliged to rely largely on the less precise method of observing with the microscope the visible effects of dyes and other reagents. This difficulty is increased by the fact that both cytoplasm and karyoplasm are not simple chemical compounds, but mixtures of many complex substances ; and both, moreover, undergo periodic changes of a complicated character which differ very widely in different kinds of cells. Our knowledge is, therefore, still fragmentary, and we have as yet scarcely passed the threshold of a subject which belongs largely to the cytology of the future.
It has been shown in the foregoing chapter that all the parts of the cell arise as local differentiations of a general protoplasmic basis. Despite the difficulties of chemical analysis referred to above, it has been determined with certainty that some at least of these organs are the seat of specific chemical change ; just as is the case in the various organs and tissues of the organism at large. Thus, the nucleus is characterized by the presence of nuclein (chromatin) which has been proved by chemical analysis to differ widely from the cytoplasmic substances, 1 while the various forms of plastids are centres for the formation of chlorophyll, starch, or pigment. These facts give ground for the conclusion that the morphological differentiation of cell-organs is in general accompanied by underlying chemical specializations which are themselves the expression of differences of metabolic activity ; and these relations, imperfectly comprehended as they are, are of fundamental importance to the student of development.
1. The Proteids and their Allies
The most important chemical compounds found in the cell are the group of protein substances, and there is every reason to believe that these form the principal basis of living protoplasm in all of its forms. These substances are complex compounds of carbon, hydrogen, nitrogen, and oxygen, often containing a small percentage of sulphur, and in some cases also phosphorus and iron. They form a very extensive group of which the different members differ considerably in physical and chemical properties, though all have certain common traits and are closely related. They are variously classified even by the latest writers. By many authors (for example Halliburton, '93) the word "proteids " is used in a broad sense as synonymous with albuminous substances, including under them the various forms of albumin (eggalbumin, cell-albumin, muscle-albumin, vegetable-albumins), globulin (fibrinogin vitellin, etc.), and the peptones (diffusible hydrated proteids). Another series of nearly related substances are the albuminoids (reckoned by some chemists among the "proteids"), examples of which are gelatin, mucin, and, according to some authors also, nuclein, and the nucleo-albumins. Some of the best authorities however, among them Kossel and Hammarsten, follow the usage of Hoppe-Seyler in restricting the word proteid to substances of greater complexity than the albumins and globulins. Examples of these are the nucleins and nucleo-proteids, which are compounds of nucleinic acid with albumin, histon, or protamin. The nucleo-proteids, found only in the nucleus, are not to be confounded with the nucleo 1 It has long been known that a form of " nuclein " may also be obtained from the nucleoalbumins of the cytoplasm, eg. from the yolk of hens' eggs (vitellin). Such nucleins differ, however, from those of nuclear origin in not yielding as cleavage-products the nuclein bases (adenin, xanthin, etc.). The term " paranuclein " (Kossel) or " pseudo-nuclein " (Hammarsten) has therefore been suggested for this substance. True nucleins containing a large percentage of albumin are distinguished as tiucU&-proteids. They may be split into albumin (or albumin radicals) and nucleinic acid, the latter yielding as cleavage-products the nuclein bases. Pseudo-nucleins containing a large percentage of albumin are designated as nucleoalbumins, which in like manner split into albumin and paranucleinic or pseudo-nucleinic acid, which yields no nuclein bases. (See Hammarsten, '94.)
332
CELL-CHEMISTRY ASD CELL^PHYSJOLOGY
albumins, which are compounds of pseudo-nucleinic acid with albumin and yield no nuclein-bases (xanthin, hypoxanthin, adenin, guanin) as decomposition products.
The distribution of these substances through the cell varies greatly not only in different cells, but at different periods in the life of the same cell. The cardinal fact always, however, remains, that there is a definite and constant contrast between nucleus and cytoplasm. The latter always contains large quantities of nucleo-albumins, certain globulins, and sometimes small quantities of albumins and peptones ; the former contains, in addition to these, nuclein and nucleo-proteids y which form its main bulk, and its most constant and characteristic feature. It is the remarkable substance, nuclein, — which is almost certainly identical with chromatin, — that chiefly claims our attention here on account of the physiological rdle of the nucleus.
2. The Nuclein Series
Nuclein was first isolated and named by Miescher, in 1 871, by subjecting cells to artificial gastric digestion. The cytoplasm is thus digested, leaving only the nuclei ; and in some cases, for instance puscells and spermatozoa, it is possible by this method to procure large quantities of nuclear substance for accurate quantitative analysis. The results of analysis show it to be a complex albuminoid substance, rich in phosphorus, for which Miescher gave the chemical formula Qd^^N^PgO^- The earlier analysis of this substance gave somewhat discordant results, as appears in the following table of percentage-compositions : 1 —
Pus-cells.
Spermatozoa of Salmon.
Human Brain.
(Hoppe-Seyler.)
(Miescher.)
(v. Jaksch.)
c
495 8
36."
50.6
H
7.IO
5- ! 5
7.6
N
15.02
13-09
1318
P
2.28
559
I.89
These differences led to the opinion, first expressed by HoppeSeylcr, and confirmed by later investigations, that there are several varieties of nuclein which form a group having certain characters in common. Altmann ('89) opened the way to an understanding of the matter by showing that " nuclein " may be split up into two substances ; namely, ( 1 ) an organic acid rich in phosphorus, to which he
1 From Halliburton, '91, p. 203. [The oxygen-percentage is omitted in this table.]
CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM 333
gave the name nuclcinic acid, and (2) a form of albumin. Moreover, the nuclein may be synthetically formed by the re-combination of these two substances. Pure nucleinic acid, for which Miescher ('96) afterward gave the formula C 40 H 54 N 14 P 4 O 27 , 1 contains no sulphur, a high percentage of phosphorus (above 9%), and no albumin. By adding it to a solution of albumin a precipitate is formed which contains sulphur, a lower percentage of phosphorus, and has the chemical characters of " nuclein. " This indicates that the discordant results in the analyses of nuclein, referred to above, were probably due to varying proportions of the two constituents; and Altmann suggested that the " nuclein " of spermatozoa, which contains no sulphur and a maximum of phosphorus, might be uncombined nucleinic acid itself. Kossel accordingly drew the conclusion, based on his own work as well as that of Liebermann, Altmann, Malfatti, and others, that "what the histologists designate as chromatin consists essentially of combinations of nucleinic acid with more or less albumin, and in some cases may even be free nucleinic acid. The less the percentage of albumin in these compounds, the nearer do their properties approach those of pure nucleinic acid, and we may assume that the percentage of albumin in the chromatin of the same nucleus may vary according to physiological conditions." 2 In the same year Halliburton, following in part Hoppe-Seyler, stated the same view as follows. The so-called " nucleins " form a series leading downward from nucleinic acid thus : —
(1) Those containing no albumin and a maximum (9-10%) of phos phorus (pure nucleinic acid). Nuclei of spermatozoa.
(2) Those containing little albumin and rich in phosphorus. Chro matin of ordinary nuclei.
(3) Those with a greater proportion of albumin — a series of sub stances in which may probably be included pyrenin (nucleoli) and plastin (linin). These graduate into
(4) Those containing a minimum (0.5 to 1%) of phosphorus —
the nucleo-albumins, which occur both in the nucleus and in the cytoplasm (vitellin, caseinogen, etc.).
Finally, we reach the globulins and albumins, especially characteristic of the cell-substance, and containing no nucleinic acid. "We thus pass by a gradual transition (from the nucleo-albumins) to the other proteid constituents of the cell, the cell-globulins, which contain no phosphorus whatever, and to the products of cell-activity, such as the proteids of serum and of egg-white, which are also principally
1 Derived from analysis of the salmon-sperm. 8 '93, p. 158.
334 CELL-CHEMISTRY AND CELL-PHYSIOLOGY
phosphorus-free." l Further, " in the processes of vital activity there are changing relations between the phosphorized constituents of the nucleus, just as in all metabolic processes there is a continual interchange, some constituents being elaborated, others breaking down into simpler products." This latter conclusion has been well established; the others, as stated by Halliburton, require some modification, on the one hand, through the results of later analyses of chromatin, on the other, because of the failure to distinguish between the nucleoproteids and the nucleo-albumins. First, it has been shown by Miescher ('96), Kossel ('96), and Mathews ('97, 2) that the chromatin of the sperm-nuclei (in fish and sea-urchins) is not pure nucleinic acid, as Altmann conjectured, but a salt of that acid, with histon, protamin, or a related substance. Thus, in the spermatozoa of the salmon, Miescher's analyses give 60.56% of nucleinic acid and 35.56% of protamin (C 16 H 28 N 9 2 ). In the herring the chromatin is a compound of nucleinic acid (over 63%) and a form of protamin called by Kossel " clupein " (C 30 H 57 N 17 O 6 ). In the sea-urchin Arbacia Mathews finds the chromatin to be a compound of nucleinic acid and " arbacin," a histon-like body. Kossel finds also that chromatin (nuclein) derived from the thymus gland, and from leucocytes, is largely a histon salt of nucleinic acid, the proportion of the latter being, however, much less than in the sperm-chromatin, while albumin is also present. In these cases, therefore, the greater part of the nucleinic acid is combined not with albumin but with a histon or protamin radical. Second, the nucleo-albumins of the cytoplasm are in no sense transitional between the nucleins and the albumins, since they contain no true nucleinic acid, but only pseudo-nucleinic acid. 2 The fact nevertheless remains that the nucleins and nucleo-proteids, though confined to the nucleus, form a series descending from such highly phosphorized bodies as the sperm-chromatin toward bodies such as the albumins, which are especially characteristic of the cytoplasm ; and that they vary in composition with varying physiological conditions. The way is thus opened for a more precise investigation of the physiological role of nucleus and cytoplasm in metabolism.
3. Staining-re action of the Nuclein Series
In bringing these facts into relation with the staining-reactions of the cell, it is necessary briefly to consider the nature of stainingreactions in general, and especially to warn the reader that in the whole field of " micro-chemistry " we are still on such uncertain ground that all general conclusions must be taken with reserve.
First, it is still uncertain how far staining-reactions depend upon chemical reaction and how far upon merely physical properties of
1, 93»P. 574- 2 C/.p. 331.
CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM 335
the bodies stained. The prevalent view that staining-reactions are due to a chemical combination of the dye with the elements of the cell has been attacked by Gierke ('85), Rawitz C97), and Fischer ('97, '99), all of whom have endeavoured to show that these reactions are of no value as a chemical test, being only a result of surfaceattraction and absorption due to purely physical qualities of the bodies stained. On the other hand, a long series of experiments, beginning with Miescher's discovery ('74) that isolated nucleinic acid forms green insoluble salts with methyl-green, and continued by Lilienfeld, Heidenhain, Paul Mayer, and others, gives strong reason to believe that beyond the physical imbibition of colour a true chemical union takes place, which, with due precautions, gives us at least a rough test of the chemical conditions existing in the cell. 1
Second, similarity of s tain ing-rcact ion is by no means always indicative of chemical similarity y as is shown, for example, by the fact that in cartilage both nuclei and inter-cellular matrix are intensely stained by methyl-green, though chemically they differ very widely.
Third, colour in itself gives no evidence of chemical nature ; for the nucleus and other elements of the same cell may be stained red, green, or blue, according to the dye employed, and to class them as " erythrophilous," "cyanophilous," and the like, is therefore absurd.
Fourth, the character of the staining-reaction is influenced and in some cases determined by the fixation or other preliminary treatment, a principle made use of practically in the operations of mordaunting, but one which may give very misleading results unless carefully controlled. Thus Rawitz ('95) shows that certain colours which ordinarily stain especially the nucleus (saffranin, gentian-violet), can be made to stain only the cytoplasm through preliminary treatment of object with solutions of tannin, followed by tartar-emetic. In like manner Mathews ('98) shows that many of the " nuclear " tar-colours (saffranin, methyl-green, etc.) stain or do not stain the cytoplasm, according as the material has been previously treated with alkaline or with acid solutions.
The results with which we now have to deal are based mainly upon experiments with tar-colours ("aniline dyes"). Ehrlich ('79) long since characterized these dyes as "acid" or "basic," according as the colouring matter plays the part of an acid or a base in the compound employed, showing further that, other things equal, the basic dyes (methyl-green, saffranin, etc.) are especially "nuclear stains" and the acid (rubin, eosin, orange, etc.) "plasma stains." Malfatti ('91), and especially Lilienfeld ('92, '93), following out Miescher's earlier work ('74), found that albumin stains preeminently in the acid stains, nucleinic acid only in the basic ; and, further, that artifi 1 Cf. Mayer, '91, '92, '97; Lilienfeld, '93; Mathews, '98.
336 CELL-CHEMISTRY AND CELL-PHYSIOLOGY
rial nucleins, prepared by combining egg-albumin with nucleinic acid in various proportions, show a varying affinity for basic and acid dyes according as the nucleinic acid is more or less completely saturated with albumin. Lilienfeld's starting-point was given by the results of Kossel's researches on the relations of the nuclein group, which are expressed as follows : l —
Nuclco-proteid (i% of P or less), by peptic digestion splits into
Peptone Nuclein (3-4% P),
by treatment with acid splits into
, ■ ,
Albumin Nucleinic acid (9-10% P),
heated with mineral acids splits into
, . _^
Phosphoric acid Nuclein bases (A carbohydrate.)
(adenin, guanin, etc.).
Now, according to Kossel and Lilienfeld, the principal nucleoproteid in the nucleus of leucocytes is nucleo-liiston, containing about 3% of phosphorus, which may be split into a form of nticlein playing the part of an acid, and an albuminoid base, the his ton of Kossel; the nuclein may in turn be split into albumin and nucleinic acid. These four substances — albumin, nucleo-histon, nuclein, nucleinic acid — thus form a series in which the proportion of phosphorus, which is a measure of the nucleinic acid, successively increases from zero to 9-10%. If the members of this series be treated with the same mixture of red acid fuchsin and basic methyl-green, the result is as follows. Albumin (egg-albumin) is stained red, nucleo-histon greenish blue, nuclein bluish green, nucleinic acid intense green. "We see, therefore, that the principle that determines the staining of the nuclear substances is always the nucleinic acid. All the nuclear substances, from those richest in albumin to those poorest in it, or containing none, assume the tone of the nuclear {i.e. basic) stain, but the combined albumin modifies the green more or less toward blue." a Lilienfeld explains the fact that chromatin in the cell-nucleus seldom appears pure green on the assumption, supported by many facts, that the proportions of nucleinic acid and albumin vary with different physiological conditions, and he suggests further that the intense staining-power of the chromosomes during mitosis is probably due to the fact that they contain a maximum of nucleinic acid. Very interesting is a comparison of the foregoing staining-reactions with those given by a mixture of a red basic dye (saffranin) and a green acid one (" light green "). With this combination an effect is given which reverses that of the Biondi-Ehrlich mixture ; i.e. the nuclein
1 From Lilienfeld, after Kossel ('92, p. 129). a Lc. t p. 394.
CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM 337
is coloured red, the albumin green, which is a beautiful demonstration of the fact that staining-reagents cannot be logically classified according to colour, but only according to their chemical nature, and gives additional ground for the view that staining-reactions of this type are the result of a chemical rather than a merely physical combination.
These results must be taken with some reserve for the following reasons: Mathews ('98) has shown that methyl-green and other basic dyes will energetically stain albumose, coagulated egg-albumin, and the cell-cytoplasm in or after treatment by alkaline fluids ; while conversely the acid dyes do not stain, or only slightly stain, these substances under the same conditions. This probably does not affect the validity of HeidenhahVs results, 1 since he worked with acid solutions. What is more to the point is the fact that hyaline cartilage and mucin, though containing no nucleinic acid, stain intensely with basic dyes. Mathews probably gives the clue to this reaction, in the suggestion that it is here probably due to the presence of other acids (in the case of cartilage a salt of chondroitin-sulphuric acid, according to Schmiedeberg); from which Mathews concludes that the basic dyes will, in acid or neutral solutions, stain any element of the tissues that contains an organic acid in a salt combination with a strong base. 2 Accepting this conclusion, we must therefore recognize that, as far as the cytoplasm is concerned, the basic or " nuclear " stains are in no sense a test for nuclein, but only for salts of organic acids in general. In case of the nucleus, however, we know from direct analysis that we are dealing with varying combinations of nucleinic acid, and hence, with the precautions indicated above, may draw provisional conditions from the staining-reactions.
Thus regarded, the changes of staining-reaction in the chromatin are of high interest. Heidenhain ('93, '94), in his beautiful studies on leucocytes, has correlated some of the foregoing results with the staining-reactions of the cell as follows. Leucocytes stained with the Biondi-Ehrlich mixture of acid fuchsin and methyl-green show the following reactions. Cytoplasm, centrosome, attraction-sphere, astral rays, and spindle-fibres are stained pure red. The nuclear substance shows a very sharp differentiation. The chromatic network and the chromosomes of the mitotic figure are green. The lininsubstance and the true nucleoli or plasmosomes appear red, like the cytoplasm. The linin-network of leucocytes is stated by Heidenhain to consist of two elements, namely, of red granules or microsomes suspended in a colourless network. The latter alone is called " linin " by Heidenhain. To the red granules is applied the term " oxychromatin," while the green substance of the ordinary chromatic network,
1 See below. 2 '98, pp. 451-452.
z
338 CELI^CHEMISTRY AND CELL-PHYSIOLOGY
forming the " chromatin " of Flemming, is called " basichromatin." * Morphologically, the granules of both kinds are exactly alike, 2 and in many cases the oxychromatin-granules are found not only in the " achromatic " nuclear network, but also intermingled with the basichroma tin-granules of the chromatic network. Collating these results with those of the physiological chemists, Heidenhain concludes that basichromatin is a substance rich in phosphorus (i.e. nucleinic acid), oxychromatin a substance poor in phosphorus, and that, further, " basichromatin and oxychromatin are by no means to be regarded as permanent unchangeable bodies but may change their colourreactions by combining with or giving off phosphorus." In other words, " the affinity of the chromatophilous microsomes of the nuclear network for basic and acid aniline dyes is regulated by certain physiological conditions of the nucleus or of the cell." 3
This conclusion, which is entirely in harmony with the statements of Kossel and Halliburton quoted above, opens up the most interesting questions regarding the periodic changes in the nucleus. The staining-power of chromatin is at a maximum when in the preparatory stages of mitosis (spireme-thread, chromosomes). During the ensuing growth of the nucleus it always diminishes, suggesting that a combination with albumin has taken place. This is illustrated in a very striking way by the history of the egg-nucleus or germinal vesicle, which exhibits the nuclear changes on a large scale. It has long been known that the chromatin of this nucleus undergoes great changes during the growth of the egg f and several observers have maintained its entire disappearance at one period. Riickert first carefully traced out the history of the chromatin in detail in the eggs of sharks, and his general results have since been confirmed by Born in the eggs of Triton. In the shark Pristinrus^ Riickert ('92, 1 ) finds that the chromosomes, which persist throughout the entire growth-period of the egg t undergo the following changes (Fig. 157): At a very early stage they are small, and stain intensely with nuclear dyes. During the growth of the egg they undergo a great increase in size, and progressively lose their staining-capacity. At the same time their surface is enormously increased by the development of long threads which grow out in every direction from the central axis (Fig. 157, A). As the egg approaches its full size, the chromosomes rapidly diminish in size, the radiating threads disappear, and the staining-capacity increases (Fig. 157, B). They are finally again reduced to minute, intensely staining bodies which enter into the equatorial plate of the first polar, mitotic figure (Fig. 157, C). How great the change of volume is may be seen from the following figures. At the beginning the chromosomes measure, at most, 12 /* (about ^uW m -) m length and
1 '94, P- 543- a/ ' c > P- 547- 8/ '» P- 548.
CHEMICAL RELATIONS OF NUCLEUS AND CYTOPLASM
339
\ p in diameter. At the height of their development they are almost eight times their original length and twenty times their original diameter. In the final period they are but 2 p in length and 1 fi in diameter. These measurements show a change of volume so enormous, even after making due allowance for the loose structure of the large chromosomes, that it cannot be accounted for by mere swelling or shrinkage. The chromosomes evidently absorb a large amount of
Pif . 157- — Chromosomes of the germinal »e drawn lo Ihe same scale. [RUCKERT.]
A. Al Ihe period ol maiimal site and min B. Later period (egg 13 mm. in diameter). C.
matter, combine with it to form a substance of diminished stainingcapacity, and finally give off matter, leaving an intensely staining substance behind. As Ruckert points out, the great increase of surface in the chromosomes is adapted to facilitate an exchange of material between the chromatin and the surrounding substance; and he concludes that the coincidence between the growth of the chromosomes and that of the egg points to an intimate connection between the nuclear activity and the formative energy of the cytoplasm.
340 CEU^CHEMISTRY AND CELL-PHYSIOLOGY
If these facts are considered in the light of the known stainingreaction of the nuclein series, we must admit that the following conclusions are something more than mere possibilities. We may infer that the original chromosomes contain a high percentage of nucleinic acid ; that their growth and loss of staining-power is due to a combination with a large amount of albuminous substance to form a lower member of the nuclein series, probably a nucleo-proteid ; that their final diminution in size and resumption of staining-power is caused by a giving up of the albumin constituent, restoring the nuclein to its original state as a preparation for division. The growth and diminished staining-capacity of the chromatin occurs during a period of intense constructive activity in the cytoplasm ; its diminution in bulk and resumption of staining-capacity coincides with the cessation of this activity. This result is in harmony with the observations of Schwarz and Zacharias on growing plant-cells, the percentage of nuclein in the nuclei of embryonic cells (meristem) being at first relatively large and diminishing as the cells increase in size. It agrees further with the fact that of all forms of nuclei those of the spermatozoa, in which growth is suspended, are richest in nucleinic acid, and in this respect stand at the opposite extreme from the nuclei of the rapidly growing egg-cell.
Accurately determined facts in this direction are still too scanty to admit of a safe generalization. They are, however, enough to indicate the probability that chromatin passes through a certain cycle in the lite of the cell, the percentage of albumin or of albumin-radicals increasing during the vegetative activity of the nucleus, decreasing in its reproductive phase./ In other words, a combination of albumin with nuclein or nucleinic acid is an accompaniment of constructive metabolism. As the cell prepares for division, the combination is dissolved and the nuclein-radicle or nucleinic acid is handed on by division to the daughter-cells. A tempting hypothesis, suggested by Mathews on the basis of Kossel's work, is that nuclein, or one of its constituent molecular groups, may in a chemical sense be regarded as the formative centre of the cell which is directly involved in the process by which food-matters are built up into the cell-substance. Could this be established, we should have not only a clear light on the changes of staining-reactions during the cycle of cell-life, but also a clue to the nuclear "control" of the cell through the process of synthetic metabolism. This hypothesis fits well with the conclusions of other physiological chemists that the nucleus is especially concerned in synthetic metabolism. Kossel concludes that the formation of new organic matter is dependent on the nucleus, 1 and that nuclein in some manner plays a leading role in this process ; and he makes
1 Schieffenlecker and Kossel, Geivebelekrg % p. 57.
PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 341
some interesting suggestions regarding the synthesis of complex organic matters in the living cell with nuclein as a starting-point. Chittenden, too, in a review of recent chemico-physiological discoveries regarding the cell, concludes : " The cell-nucleus may be looked upon as in some manner standing in close relation to those processes which have to do with the formation of organic substances. Whatever other functions it may possess, it evidently, through the inherent qualities of the bodies entering into its composition, has a controlling power over the metabolic processes in the cell, modifying and regulating the nutritional changes " C94).
These conclusions, in their turn, are in harmony with the hypothesis advanced twenty years ago by Claude Bernard ('78), who maintained that the cytoplasm is the seat of destructive metabolism, the nucleus the organ of constructive metabolism and organic synthesis, and insisted that the rdlc of the nucleus in nutrition gives the key to its significance as the organ of development, regeneration, and inheritance. 1
B. Physiological Relations of Nucleus and Cytoplasm
How nearly the foregoing facts bear on the problem of the morphological formative power of the cell is obvious ; and they have in a measure anticipated certain conclusions regarding the rdle of nucleus and cytoplasm, which we may now examine from a somewhat different point of view.
Briicke long ago drew a clear distinction between the chemical and molecular composition of organic substances, on the one hand, and, on the other hand, their definite grouping in the cell by which arises organization in a morphological sense. Claude Bernard, in like manner, distinguished between chemical synthesis, through which organic matters are formed, and morphological synthesis, by which they are built into a specifically organized fabric ; but he insisted that these two processes are but different phases or degrees of the same phenomenon, and that both are expressions of the nuclear activity. We have now to consider some of the evidence that the power of morphological, as well as of chemical, synthesis centres in the nucleus, and that this is therefore to be regarded as the especial organ of inheritance. This evidence is mainly derived from the comparison of nucleated and non-nucleated masses of protoplasm ; from the form,
1 " II scmble done que la cellule qui a perdu son noyau soit sterilisee au point de vue de la generation, e'est a dire de la synthese morphologique, et qu'elle le soit aussi au point de vue de la synthese chimique, car elle cesse de produire des principes i m media ts, et ne peut gucrc qu'oxyder et detruire ceux qui s'y etaicnt accumules par une elaboration anterieure du noyau. II semble done que le noyau soit le germe de nutrition dela cellule ; il attire autour de lui et llabore les materiaux nutritifs" ('78, p. 523).
34^ CELL-CHEMISTRY AND CELL-PHYSIOLOGY
position, and movements of the nucleus in actively growing or metabolizing cells; and from the history of the nucleus in mitotic celldivision, in fertilization, and in maturation.
I. Experiments on Unicellular Orgt
Brandt ('77) long since observed that enucleated fragments of Aetinosphcerium soon die, while nucleated fragments heal their wounds and continue to live. The first decisive comparison between nucleated and non-nucleated masses of protoplasm was, however, made by Moritz Nussbaumin 1884 in the case of an infusorian, Oxytruha. If one of these animals be cut into two pieces, the subsequent behaviour of the two fragments depends on the presence or absence of the nucleus or a nuclear fragment. The nucleated fragments quickly heal the wound, regenerate the missing portions, and thus produce a perfect animal. On the other hand, enucleated fragments, consisting of cytoplasm only, quickly perish. Nussbaum therefore drew the conclusion that the nucleus is indispensable for the formative energy of the cell. The experiment was soon after repeated by Gruber('S5) in the case of Stenlor, another infusorian, and with the same result (Fig. 159). Fragments possessing a large fragment of the nucleus completely regenerated within twenty-four hours. If the nuclear fragment were smaller, the regeneration proceeded more slowly. If no nuclear substance were present, no regeneration took place, though the wound closed and the fragment lived for a considerable time. The only exception — but it is a very significant one — was the case of individuals in which the process of normal fission had begun ; in these a non-nucleated fragment in which the formation of a new peristome had already been initiated healed the wound and completed the formation of the peri
imal. showing planes o(
The middle piec.
. The enucleated pieces.
the right, swim ab
out for a time, but finally
PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 343
stome. Lillie ('96) has recently found that Stentor may by shaking be broken into fragments of all sizes, and that nucleated fragments as small as 5 \ the volume of the entire animal are still capable of complete regeneration. All non-nucleated fragments perish.
These studies of Nussbaum and Gruber formed a prelude to more extended investigations in the same direction by Gruber, Balbiani, Hofer, and especially Verworn Verworn ('88) proved that in Polystomella, one of the Foraminifera, nucleated fragments are able to
Fig. 159. — Regeneration tn the unicellular A. Animal divided into three pieces, each
Stentor, [From GRUBER after Balbiani.]
ining a fragment of the nucleus. B. The three
The three fragments after twentjr-Iour hours, each regenerated
repair the shell, while non-nucleated fragments lack this power. Balbiani ('89) showed that although non-nucleated fragments of Infusoria had no power of regeneration, they might nevertheless continue to live and swim actively about for many days after the operation, the contractile vacuole pulsating as usual. Hofer ('89), experimenting on Amccba, found that non-nucleated fragments might live as long as fourteen days after the operation (Fig. 160). Their movements continued, but were somewhat modified, and little by little ceased, but the pulsations of the contractile vacuole were but slightly affected ; they lost more or less completely the capacity to
344
CELL-CHEMISTRY AND CELL-PHYSIOLOGY
digest food, and the power of adhering to the substratum. Nearly at the same time Vcrworn ('89) published the results of an extended comparative investigation of various Protozoa that placed the whole matter in a very clear light. His experiments, while fully confirming the accounts of his predecessors in regard to regeneration, added many extremely important and significant results. Non-nucleated fragments both of Infusoria {e.g. Lachrymaria) and rhizopods {Poly
[HOFER.]
after the opera
stomella, Thalassicolla) not only live for a considerable period, but perform perfectly normal and characteristic movements, show the same susceptibility to stimulus, and have the same power of ingulfing food, as the nucleated fragments. They lack, however, the power of digestion and secretion. Ingested food-matters may be slightly altered, but are never completely digested. The non-nucleated fragments are unable to secrete the material for a new shell {Polysto
PHYSIOLOGICAL JtELA TtONS OF NUCLEUS AND CYTOPLASM 345
tnella) or the slime by which the animals adhere to the substratum {Amceba, Difflugia, Polystomdla). Beside these results should be placed the well-known fact that dissevered nerve-fibres in the higher animals are only regenerated from that end which remains in connection with the nerve-cell, while the remaining portion invariably degenerates.
Ml
1 1
^
Formation of membranes by protoplasmi of Cucurbits, ihowirif
ts of plasmolyzed cells. [TOWN
Fig. 161 .
A. Plasmol»ied cell, leaf-h; 8. Calyx-hair 'of GailUrdia ; nucleated fra C. Rool-hair of Miinhanlia ; all Ihc fragmei
wiih nucleated fragment of adjoining cell.
These beautiful observations prove that destructive metabolism, as manifested by coordinated forms of protoplasmic contractility, may go on for some time undisturbed in a mass of cytoplasm deprived of a nucleus. On the other hand, the building up of new chemical or morphological products by the cytoplasm is only initiated in the presence of a nucleus and soon ceases in its absence. These facts form a complete demonstration that the nucleus plays an essential
346 CELlr- CHEMISTRY AND CELL- PHYSIOLOGY
part not only in the operations of synthetic metabolism or chemical synthesis, but also in the morphological determination of these operations, i.e. the morphological synthesis of Bernard — a point of capital importance for the theory of inheritance, as will appear beyond.
Convincing experiments of the same character and leading to the same result have been made on the cells of plants. Francis Darwin (J77) observed more than twenty years ago that movements actively continued in protoplasmic filaments, extruded from the leaf -hairs of Dipsacus, that were completely severed from the body of the cell. Conversely, Klebs ('79) soon afterward showed that naked protoplasmic fragments of Vaucheria and other algae were incapable of forming a new cellulose membrane if devoid of a nucleus ; and he afterward showed ('87) that the same is true of Zygnema and CEdogonium. By plasmolysis the cells of these forms may be broken up into fragments, both nucleated and non-nucleated. The former surround themselves with a new wall, grow, and develop into complete plants ; the latter, while able to form starch by means of the chlorophyll they contain, are incapable of utilizing it, and are devoid of the power of forming a new membrane, and of growth and regeneration. A beautiful confirmation of this is given by Townsend ('97), who finds in the case of root-hairs and pollen-tubes, that when the protoplasm is thus broken up, a membrane may be formed by both nucleated and non-nucleated fragments, by the latter however only when they remain connected with the nucleated masses by protoplasmic strands, however fine. If these strands be broken, the membrane-forming power is lost. Of very great interest is the further observation (made on leafhairs in Cucurbita) that the influence of the nucleus may thus extend from cell to cell, an enucleated fragment of one cell having the power to form a membrane if connected by intercellular bridges with a nucleated fragment of an adjoining cell (Fig. 161).
2. Position and Movements of the Nucleus
Many observers have approached the same problem from a different direction by considering the position, movements, and changes of form in the nucleus with regard to the formative activities in the cytoplasm. To review these researches in full would be impossible, and we must be content to consider only the well-known researches of Haberlandt C77) and Korschelt ('89), both of whom have given extensive reviews of the entire subject in this regard. Haberlandt's studies related to the position of the nucleus in plant-cells with especial regard to the growth of the cellulose membrane. He determined the very significant fact that local growth of the cell-wall is always preceded by a movement of the nucleus to the point of growth. Thus, in the formation of epidermal cells, the nucleus lies at first near
PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 347
the centre, but as the outer wall thickens, the nucleus moves toward it, and remains closely applied to it throughout its growth, after which the nucleus often moves into another part of the cell (Fig. 162, A, B). That this is not due simply to a movement of the nucleus toward the air and light is beautifully shown in the coats of certain seeds, where the nucleus moves, not to the outer, but to the inner wall of the cell, and here the thickening takes place (Fig. 162, C), The same position
Pig. 162. — Position of the nuclei in growing plant-cells. [Haberlandt.]
A. Young epidermal cell of Luzula with central nucleus, before thickening of the membrane. B. Three epidermal cells of Afonstera, during the thickening of the outer wall. C. Cell from the seed-coat of Scopulina, during the thickening of the inner wall. D. E. Position of the nuclei during the formation of branches in the root-hairs of the pea.
of the nucleus is shown in the thickening of the walls of the guardcells of stomata, in the formation of the peristome of mosses, and in many other cases. In the formation of root-hairs in the pea, the primary outgrowth always takes place from the immediate neighbourhood of the nucleus, which is carried outward and remains near the tip of the growing hair (Fig. 162, D, E). The same is true of the rhizoids of fern-prothallia and liverworts. In the hairs of aerial plants this
348 CELL-CHEMISTRY AND CELL-PHYSIOLOGY
rule is reversed, the nucleus lying near the base of the hair ; but this apparent exception proves the rule, for both Hunter and Haberlandt show that in this case growth of the hair is not apical, but proceeds from the base ! Very interesting is Haberlandt's observation that in the regeneration of fragments of Vaiicheria the growing region, where a new membrane is formed, contains no chlorophyll, but numerous nuclei. The general result, based on the study of a large number of cases, is, in Haberlandt's words, that " the nucleus is in most cases placed in the neighbourhood, more or less immediate, of the points at which growth is most active and continues longest." This fact points to the conclusion that " its function is especially connected with the developmental processes of the cell," l and that "in the growth of the cell, more especially in the growth of the cell-wall, the nucleus plays a definite part."
Korschelt's work deals especially with the correlation between form and position of the nucleus and the nutrition of the cell, and since it bears more directly on chemical than on morphological synthesis, may be only briefly reviewed at this point. His general conclusion is that there is a definite correlation, on the one hand, between the position of the nucleus and the source of food-supply, on the other hand, between the size of the nucleus and the extent of its surface and the elaboration of material by the cell. In support of the latter conclusion many cases are brought forward of secreting cells in which the nucleus is of enormous size and has a complex branching form. Such nuclei occur, for example, in the silk-glands of various lepidopterous larvae (Meckel, Zaddach, etc.), which are characterized by an intense secretory activity concentrated into a very short period. Here the nucleus forms a labyrinthine network (Fig. 14, E\ by which its surface is brought to a maximum, pointing to an active exchange of material between nucleus and cytoplasm. The same type of nucleus occurs in the Malpighian tubules of insects (Leydig, R. Hertwig), in the spinning-glands of amphipods (Mayer), and especially in the nutritive cells of the insect ovary already referred to at page 151. Here the developing ovum is accompanied and surrounded by cells, which there is good reason to believe are concerned with the elaboration of food for the egg-cell. In the earwig Forficula each egg is accompanied by a single large nutritive cell (Fig. 163), which has a very large nucleus rich in chromatin (Korschelt). This cell increases in size as the ovum grows, and its nucleus assumes the complex branching form shown in the figure. In the butterfly Vanessa there is a group of such cells at one pole of the cgg t from which the latter is believed to draw its nutriment (Fig. 77). A very interesting case is that of the annelid Ophryotrocha, referred to at page 151. Here, as described by Korschelt, the egg floats
1 I.e., p. 99.
PHYSIOLOGICAL DELATIONS OF NUCLEUS AND CYTOPLASM
\
V
in the perivisceral fluid, accompanied by a nurse-cell having a very large chromatic nucleus, while that of the egg is smaller and poorer inchromatin. Astheegg com pletes its growth, the nurse-cell dwindles away and finally perishes (Fig, 76). In all these cases it is scarcely possible to doubt that the egg is in a measure relieved of the task of elaborating cytoplasmic products by the nurse-cell, and that the great development of the nucleus in the latter is correlated with this function.
Regarding the position and movements of the nucleus, Korschelt reviews many facts pointing toward the same conclusion. Perhaps the most suggestive of these relate to the nucleus of the egg during its ovarian history. In many of the insects, as in both the juala,
CaSCS referred to above. Below, a portion of ihe nearly ripe egg (*>. showing deuio the e ge .„ud« u , a. first SSSSSSSH^iSiJS^'^m
occupies a central pOSl- cessively younger stages of egg and nurse are shown above.
tion, but as the egg begins to grow, it moves to the periphery on the side turned toward the nutritive cells. The same is true in the ovarian eggs of some other animals, good examples of which are afforded by various ccelcnterates, e.g. in medusae (Claus, Hertwig) and actinians (Korschelt, Hertwig), where the germinal vesicle is always near the point of attachment of the egg. Most suggestive of all is the case of the water-beetle Dytisctis, in which Korschelt was able to observe the movements and changes of form in the living object. The eggs here lie in a single series alternating with chambers of nutritive cells. The latter contain granules which are believed by Korschelt to pass into the egg, perhaps bodily, perhaps by dissolving and entering in a liquid form. At all events,
S V -,
Fig. 163. — Upper port]
350 CELL-CHEMISTRY AND CELL-PHYSIOLOGY
•
the egg contains accumulations of similar granules, which extend inward in dense masses from the nutritive cells to the germinal vesicle, which they may more or less completely surround. The latter meanwhile becomes amoeboid, sending out long pseudopodia, which are always directed toward the principal mass of granules (Fig. jj\ The granules could not be traced into the nucleus, but the latter grows rapidly during these changes, proving that matter must be absorbed by it, probably in a liquid form. 1
Among other facts pointing in the same direction may be mentioned Miss Huie's ('97) observations on the gland-cells of Drosera, and those of Mathews ('99) on the changes of the pancreas-cell in Necturus. Stimulus of the gland-cells in the leaf of Drosera causes a rapid exhaustion and change of staining-capacity in the cytoplasm. During the ensuing repose the cytoplasm is rebuilt out of material laid down immediately around the nucleus, and agreeing closely in appearance and staining-reaction with the achromatic nuclear constituents. The chromatin increases in bulk during a period preceding the constructive phase, but decreases (while the nucleolar material increases) as the cytoplasm is restored. In the pancreas-cell, as has long been known, the " loaded " cell (before secretion) is filled with metaplasmic zymogen-granules, which disappear during secretion, the cell meanwhile becoming filled with protoplasmic fibrils (Fig. 18). During the ensuing period of " rest " the zymogen-granules are re-formed at the expense of the fibrillar material, which is finally found only at the base of the cell near the nucleus. Upon discharge of the secretion (granule-material) the fibrillae again advance from the nucleus toward the periphery. Mathews shows that many if not all of them may be traced at one end actually into the nuclear wall, and concludes that they are directly formed by the nucleus.
Beside the foregoing facts may be placed the strong evidence reviewed at pages 156-158, indicating the formation of the yolk-nucleus, and indirectly of the yolk-material, by the nucleus. All of these and a large number of other observations in the same direction lead to the conclusion that the cell-nucleus plays an active part in nutrition, and that it is especially active during the constructive phases. On the whole, therefore, the behaviour of the nucleus in this regard is in harmony with the result reached by experiment on the one-celled forms, though it gives in itself a far less certain and convincing result. 2
1 Mention may conveniently here be made of Richard Hertwig's interesting observation that in starved individuals of Actinosplunium the chromatin condenses into a single mass, while in richly fed animals it is divided into fine granules scattered through the nucleus ('98, p. 8).
2 Loeb ('98, '99) makes the interesting suggestion that the nucleus is especially concerned in the oxydative processes of the cell, and that this is the key to its r6U in the synthetic process. It has been shown that oxydations in the living tissues are probably
PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 351
We now turn to evidence which, though less direct than the above, is scarcely less convincing. This evidence, which has been exhaustively discussed by Hertwig, Weismann, and Strasburger, is drawn from the history of the nucleus in mitosis, fertilization, and maturation. It calls for only a brief review here, since the facts have been fully described in earlier chapters.
3. The Nucleus in Mitosis
To Wilhelm Roux ('83) we owe the first clear recognition of the fact that the transformation of the chromatic substance during mitotic division is manifestly designed to effect a precise division of all its parts, — i.e. a panmeristic division as opposed to a mere mass-division, — and their definite distribution to the daughter-cells. "The essential operation of nuclear division is the division of the mother-granules " {i.e. the individual chromatin-grains) ; " all the other phenomena are for the purpose of transporting the daughter-granules derived from the division of a mother-granule, one to the centre of one of the daughter-cells, the other to the centre of the other." In this respect the nucleus stands in marked contrast to the cytoplasm, which undergoes on the whole a mass-division, although certain of its elements, such as the plastids and the centrosome, may separately divide, like the elements of the nucleus. From this fact Roux argued, first, that different regions of the nuclear substance must represent different qualities, and second, that the apparatus of mitosis is designed to distribute these qualities, according to a definite law, to the daughtercells. The particular form in which Roux and Weismann developed this conception has now been generally rejected, and in any form it has some serious difficulties in its way. We cannot assume a precise localization of chromatin-elements in all parts of the nucleus ; for on the one hand a large part of the chromatin may degenerate or be cast out (as in the maturation of the egg), and on the other hand in the Protozoa a small fragment of the nucleus is able to regenerate the whole. Nevertheless, the essential fact remains, as Hertwig, Kolliker, Strasburger, De Vries, and many others have insisted, that in mitotic cell-division the chromatin of the mother-cell is distributed with the most scrupulous equality to the nuclei of the daughter-cells, and that in this regard there is a most remarkable contrast between nucleus and cytoplasm. This holds true with such wonderful constancy
dependent upon certain substances (oxydation ferments) that in some manner, not yet clearly understood, facilitate the process; and the work of Spitzer ('97) has shown that these substances (obtained from tissue-extracts) belong to the group of nucleo-proteids, which are characteristic nuclear substances. The view thus suggested opens a further way toward more exact inquiry into the nuclear functions, though it is not to be supposed that the nucleus is the sole oxydative centre of the cell, as is obvious from the prolonged activity of non-nucleated protoplasmic masses.
352
CELL-CHEMISTRY AND CELL-PHYSIOLOGY
throughout the series of living forms, from the lowest to the highest, that it must have a deep significance. And while we are not yet in a position to grasp its full meaning, this contrast points unmistakably to the conclusion that the most essential material handed on by the mother-cell to its progeny is the chromatin, and that this substance therefore has a special significance in inheritance.
4. The Nucleus in Fertilization
The foregoing argument receives an overwhelming reenforcement
from the facts of fertilization.
Although the ovum supplies nearly all the cytoplasm for the embryonic body, and the spermatozoon at most only a trace, the latter is nevertheless as potent in its effect on the offspring as the former. On the other hand, the nuclei contributed by the germ-c^lls, though apparently different, become tn the end exactly equivalent in every visible respect — in structure, in staining-reactions, and in the number and form of the chromosomes to which each gives rise. But furthermore the substance of the two germ-nuclei is distributed with absolute equality, certainly to the first two cells of the embryo, and probably to all later-formed cells. The latter conclusion, which long remained a mere surmise, has been rendered nearly a certainty by the remarkable observations of Riickert, Zoja, and Hacker, described in Chapters IV. and VI. We must therefore accept the high probability of the conclusion that the specific character of the ceil is in the last analysis determined by that of the nucleus, that is by the chromatin, and that in the equal distribution of paternal and maternal chromatin to all the cells of the offspring we find the physiological explanation of the fact that
PHYSIOLOGICAL RELATIONS OF NUCLEUS AND CYTOPLASM 353
every part of the latter may show the characteristics of either or both parents.
Boveri ('89, '95, 1) has attempted to test this conclusion by a most ingenious and beautiful experiment ; and although his conclusions do not rest on absolutely certain ground, they at least open the way to a decisive test. The Hertwig brothers showed that the eggs of seaurchins might be broken into pieces by shaking, and that spermatozoa would enter the enucleated fragments and cause them to segment. Boveri proved that such a fragment, if fertilized by a spermatozoon, would even give rise to a dwarf larva, indistinguishable from the normal in general appearance except in size. The nuclei of such larvae are considerably smaller than those of the normal larvae, and were shown by Morgan ('95, 4) to contain only half the number of chromosomes, thus demonstrating their origin from a single sperm-nucleus. Now, by fertilizing enucleated egg-fragments of one species {Sphcerechinus granulans) with the spermatozoa of .another {Echinus microtuberculatus), Boveri obtained in a few instances dwarf Plutei showing except in size the pure paternal characters {i.e. those of Echinus, Fig. 164). From this he concluded that the maternal cytoplasm has no determining effect on the offspring, but supplies only the material in which the sperm-nucleus operates. Inheritance is, therefore, effected by the nucleus alone.
The later studies of Seeliger ('94), Morgan ('95, 4), and Drisch ('98, 3) showed that this result is not entirely conclusive, since hybrid larvae arising by the fertilization of an entire ovum of one species by a spermatozoon of the other show a very considerable range of variation ; and while most such hybrids are intermediate in character between the two species, some individuals may nearly approximate to the characters of the father or the mother. Despite this fact Boveri ('95, 1) has strongly defended his conclusion, though admitting that only further research can definitely decide the question. It is to be hoped that this highly ingenious experiment may be repeated on other forms which may afford a decisive result.
5. The Nucleus in Maturation
Scarcely less convincing, finally, is the contrast between nucleus and cytoplasm in the maturation of the germ-cells. It is scarcely an exaggeration to say that the whole process of maturation, in its broadest sense, renders the cytoplasm of the germ-cells as unlike, the nuclei as like, as possible. The latter undergo a series of complicated changes which result in a perfect equivalence between them at the time of their union, and, more remotely, a perfect equality of distribution to the embryonic cells. The cytoplasm, on the other
2 A
354 CELL-CHEMISTRY AND CELL-PHYSIOLOGY
hand, undergoes a special differentiation in each to effect a secondary division of labour between the germ-cells. When this is correlated with the fact that the germ-cells, on the whole, have an equal effect on the specific character of the embryo, we are again forced to the conclusion that this effect must primarily be sought in the nucleus, and that the cytoplasm is in a sense only its agent.
C. The Centrosome
Existing views regarding the functions of the centrosome may conveniently be arranged in two general groups, the first including those which regard this structure as a relatively passive body, the second those which assume it to be an active organ. To the first belongs the hypothesis of Heidenhain C94), accepted by Kostanecki ('97, 1) and some others, that the centrosome serves essentially as an insertionpoint for the astral rays ("organic radii"), and plays a relatively passive part in the phenomena of mitosis, the active functions being mainly performed by the surrounding structures. To the same category belongs the view of Miss Foot that the formation of the centrosome is, as it were, incidental to that of the aster — "the expression, rather than the cause, of cell-activity " ('97, p. 810). To the second group belong the views of Van Beneden, Boveri, Butschli, Carnoy, and others who regard the centrosome as playing a more active r6le in the life of the cell. Both of the former authors have assumed the centrosomes to be active centres by the action of which the astral systems arc organized ; and they are thus led to the conclusion that the centrosome is essentially an organ for cell-division and fertilization (Boveri), and in this sense is the "dynamic centre" of the cell. 1 To Carnoy and Butschli is due the interesting suggestion 2 that the centrosomes are to be regarded further as centres of chemical action to which their remarkable effect on the cytoplasm is due. That the centrosome is an active centre, rather than a passive body or one created by the aster-formation, is. strongly indicated by its behaviour both in mitosis and in fertilization. Griffin ('96, '99) points out that at the close of division in Thalassema the daughter-centrosomes migrate away from the old astral centre and incite about themselves in a different region the new astral systems for the ensuing mitosis (Figs. 99, 155); and similar conditions are described by Coe in Ccrcbratulus ('98). In fertilization the aster-formation cannot be regarded as a general action of the cytoplasm, but as a local one due to a local stimulus given by something in the spermatozoon ; for in polyspermy a sperm-aster is formed for every spermatozoon (p. 198). This stimulus is given by something in the middle 1 ( 7- PP- 7 6 » J 2 - 2 0^ P- IIO «
THE CENTROSOME 355
piece (p. 212), which is itself genetically related to the centrosome of the last cell- generation (p. 1 70). These facts seem explicable only under the assumption that in these cases the centrosome, or a substance which it carries, gives an active stimulus to the cytoplasm which incites the aster-formation about itself, and in the words of Griffin " disengages the forces at work in mitosis " ('96, p. 174). For these reasons I incline to the view that in the artificial aster-formation described by Morgan 1 the centrosomes there observed should not be regarded as the creations of the asters, but rather as local deposits of material which incite the aster-formation around them. That the centrosomes or astral centres are centres of division (whether active or passive) is beautifully shown by Boveri's interesting observations on " partial fertilization " referred to at page 194.
Again, Boveri has observed that the segmenting ovum of Ascaris sometimes contains a supernumerary centrosome that does not enter
Fig. 165. — liggs of Ascata with supernumerary cenlrosome. [BoVI A. First cleavage-spindle above, isolated centrosome below. B. Result of the ensuing division.
into connection with the chromosomes, but lies alone in the cytoplasm (Fig. 165). Such a centrosome forms an independent centre of division, the cell dividing into three parts, two of which are normal blastomeres, while the third contains only the centrosome and attraction-sphere. The fate of such eggs was not determined, but they form a complete demonstration that it is in this case the centrosome and not the nucleus that determines the centres of division in the cell-body. Scarcely less conclusive is the case of dispermic eggs in sea-urchins. In such eggs both sperm-nuclei conjugate with the eggnucleus, and both sperm-centrosomes divide (Fig. t66). The cleavage-nucleus, therefore, arises by the union of three nuclei and four centrosomes. Such eggs divide at the first cleavage into four equal blastomeres, each of which receives one of the centrosomes. 1 Cf. P . 307.
356
CELL-CHEMISTRY AND CELL-PHYSIOLOGY
The latter must, therefore, be the centres of division ; * though it must not be forgotten that, in some cases at any rate, normal division requires the presence of nuclear matter (p. 108).
The centrosome must, however, be something more than a mere division-centre ; for, on the one hand, in leucocytes and pigment-cells the astral system formed about it is devoted, as there is good reason to believe, not to cell-division, but to movements of the cell-body as a whole; and, on the other hand, as we have seen (pp. 165, 172), it is concerned in the formation of the flagella of the spermatozoa and spermatozoids, and probably also in that of cilia in epithelial cells. Strasburger ('97) was thus led to the conclusion that the centrosome is essentially a mass of kinoftlasm, i.e. the active motor plasm, 2 and a nearly similar view has been adopted by several recent zoologists.
A B C
Fig. 166. — Cleavage of dispermic egg of Tqxopncustes.
A. One sperm-nucleus has united with the egg-nucleus, shown at a. b. ; the other lies above. Both sperm-asters have divided to form amphiasters (a. b. and c. d.). B. The cleavage-nucieus, formed by union of the three germ-nuclei, is surrounded by the four asters. C. Result of the first cleavage, the four blastomeres lettered to correspond with the four asters.
Henneguy concludes that the centrosomes are " motor centres of the kinoplasm " both for external and for internal manifestations. 8 Len'hoss£k regards them as " motors " for the control of ciliary action as well as for that of the spermatozoon, 4 and perhaps also for that of muscle-fibriHae. 6 Zimmerman concludes that "the microcentrum is the motor centre of the cell, that is, the * kinocentrum ' opposed to the nucleus as the 'chemocentrum.' " 8 Regarding their control of ciliary action, he makes the same suggestion as that of Henneguy and Lenhoss^k cited above. He adds the further very interesting suggestions that the centrosomes may be concerned with the pseudopodial movements in the epithelial cells of the intestine, and that they may
1 This phenomenon was first observed by Hertwig, and afterward by Driesch. I have repeatedly observed the internal changes in the living eggs of Toxopneustes.
2 Cf. p. 221. * '98, p. 107. • '98, p. 697.
8 '98, p. 495
6 '
99, p. 342.
THE CENTROSOME 357
also be concerned in the protoplasmic contraction of gland-cells by which the excretion is expelled- [This is based on the fact that the centrosomes are found in the free (pseudopodia- forming) ends of the epithelial cells, and on the position of the centrosomes in gobletcells (Fig. 23) and in those of the lachrymal gland.] Peter ('99) has attempted to test these conclusions experimentally by cutting or tearing off cilia from the cell-body (gut-epithelium of Anodonta) and also by isolating the tails of spermatozoa. In groups consisting of only a few cilia, separated from the nucleus, the movements actively continue, while those that are separated from the basal bodies cease to beat. Spermatozoon tails separated from the head also continue to
Pig. 1*7. — Omirosomra
crfly. [HENNEGUV.]
move, but only if they remain connected with the middle-piece. Peter, therefore, supports the above conclusions of Henneguy and Lenhossek. On the other hand, Meves ('99) finds that movements of the undulating membrane in the tails of salamander-spermatozoa continue if the middle-piece be entirely removed ; white a number of earlier observers' have observed in flagellates that a flagellum separated from the body may actively continue its movements for a considerable time.
Further research is therefore required to test these suggestions.
The intimate connection of the centrosomes with the formation, on the
one hand, of the astral rays, on the other of contractile organs, such
> See Klebs, 'S3, Bfitichli, '85, Fischer, '94, 3.
358 CELL-CHEMISTRY AND CELL-PHYSIOLOGY
as cilia, flagella, and pseudopodia, 1 the centrosomes in ciliated cells and spermatozoa, and in the swarm-spores of Noctiluca, is, however, a most striking fact, and is one of the strongest indirect arguments in favour of the general theory of fibrillar contractility in mitosis.
D. Summary and Conclusion
The facts reviewed in the foregoing pages converge to the conclusion that the differentiation of the cell-substance into nucleus and cytoplasm is the expression of a fundamental physiological division of labour in the cell. Experiments upon unicellular forms demonstrate that, in the entire absence of a nucleus, protoplasm is able for a considerable time to liberate energy and to manifest coordinated activities dependent on destructive metabolism. There is here substantial ground for the view that the cytoplasm is the principal seat of these activities in the normal cell. On the other hand, there is strong cumulative evidence that the nucleus is intimately concerned in the constructive or synthetic processes, whether chemical or morphological.
That the nucleus has such a significance in synthetic metabolism is proved by the fact that digestion and absorption of food and growth soon cease with its removal from the cytoplasm, while destructive metabolism may long continue as manifested by the phenomena of irritability and contractility. It is indicated by the position and movements of the nucleus in relation to the food-supply and to the formation of specific cytoplasmic products. It harmonizes with the fact, now universally admitted, that active exchanges of material go on between nucleus and cytoplasm. The periodic changes of staining-capacity undergone by the chromatin during the cycle of celllife, taken in connection with the researches of physiological chemists on the chemical composition and staining-reactions of the nuclein series, indicate that the phosphorus-rich substance known as nuclcinic acid plays a leading part in the constructive process. During the vegetative phases of the cell this substance is combined with a large amount of the albumin radicles histon, protamin, and related substances, and probably in part with albumin itself, to form nuclein. During the mitotic or reproductive processes this combination appears to be dissolved, the albuminous elements being in large part split off, leaving the substance of the chromosomes with a high percentage of nucleinic acid, as is shown by direct analysis of the sperm-nucleus and is indicated by the staining-reactions of the chromosomes. There is, therefore, considerable ground for the hypothesis that in a chemical sense this substance is the most essential nuclear element handed on from cell to cell, whether by cell-division or by fertilization ; and that it may be a primary factor in the constructive processes of the nucleus and through these be indirectly concerned with those of the cytoplasm.
1 Cf' PP* 9 2 » I02 » on the central granule of the Heliozoa.
The role of the nucleus in constructive metabolism is intimately related with its rdle in morphological synthesis, and thus in inheritance ; for the recurrence of similar morphological characters must in the last analysis be due to the recurrence of corresponding forms of metabolic action of which they are the outward expression. That the nucleus is in fact a primary factor in morphological as well as chemical synthesis is demonstrated by experiments on unicellular plants and animals, which prove that the power of regenerating lost parts disappears with its removal, though the enucleated fragment may continue to live and move for a considerable period. That the nuclear substance, and especially the chromatin, is a leading factor in inheritance is powerfully supported by the facts of maturation, fertilization, and cell-division. In maturation the germ-nuclei are by an elaborate process prepared for the subsequent union of equivalent chromatic elements from the two sexes. By fertilization these elements are brought together, and by mitotic division distributed with exact equality to the embryonic cells. The result, which is especially striking in the case of hybrid-fertilization, proves that the spermatozoon is as potent in inheiitance as the ovum, though the latter contributes an amount of cytoplasm which is but an infinitesimal fraction of that supplied by the ovum.
It remains to be seen whether the chromatin can actually be regarded as the idioplasm or physical basis of inheritance, as maintained by Hertwig and Strasburger. Verworn has justly urged that the nucleus cannot be regarded as the sole vehicle of inheritance, since the cooperation of both nucleus and cytoplasm is essential to complete cell-life ; and, as will be shown in Chapter IX., the cytoplasmic organization plays an important rdle in shaping the course of development. Considered in all their bearings, however, the facts seem to accord best with the hypothesis that the cytoplasmic organization is itself determined, in the last analysis, by the nucleus ; l and the principle for which Hertwig and Strasburger contended is thus sustained.
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Phys., XLI. 1892. Id. — Allgemeine Physiologic Jena, 1895. Whitman, C. 0. — The Seat of Formative and Regenerative Energy : Journ. Aforph.,
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Wilson EB. The Cell in Development and Inheritance. Second edition (1900) New York, 1900.
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